Kouhei Inamura

Laminar distribution of pheromone‐receptive neurons in rat vomeronasal epithelium

1 Responses of vomeronasal sensory neurons to urine excreted from rats, mice and hamsters were studied by the on‐cell patch clamp method in slices of sensory epithelium from female Wistar rats. 2 The urine excreted from male and female Wistar rats, male Donryu rats and male C57BL/6 mice induced relatively large responses, while urine from male Sprague‐Dawley rats and male Syrian hamsters induced small responses. 3 Of the 62 neurons responding to urine, 57 responded to only one of the urine preparations. 4 The sensory neurons that responded to the male Wistar urine were localized in the apical position of the epithelium where one type of GTP‐binding protein, Gi2α, is selectively expressed. The neurons in the basal position of the epithelium, which express Goα, responded to urine from the other animals. 5 This study demonstrates that sensory neurons responsive to different urinary pheromones are localized in a segregated layer in the rat vomeronasal sensory epithelium.

Regionalization of Fos immunostaining in rat accessory olfactory bulb when the vomeronasal organ was exposed to urine

The distribution of Fos‐immunoreactive (Fos‐ir) cells in the accessory olfactory bulb (AOB) of rats following vomeronasal organ exposure to urine was studied. Following exposure to male and female Wistar rat urine, Fos‐ir cells were found in the mitral/tufted cell layer, granule cell layer and periglomerular cell layer of the AOB of female Wistar rat, with the highest number in the granule cell layer. Exposure to water or removal of the vomeronasal organ suppressed the expression of Fos‐ir cells. These results suggest that female Wistar rats specifically detect urinary substances derived from male or female Wistar rats via the vomeronasal organ. Exposure of the vomeronasal organ of female Wistar rats to male Wistar urine induced the appearance of many more Fos‐ir cells in all layers of the AOB than exposure to female Wistar urine. As for the mitral/tufted cell layer, the density of Fos‐ir cells in the rostral portion (Gi2α‐positive) of all regions of the AOB was about twice as high as that in the caudal portion when male urine was given. The distribution pattern of Fos‐ir cells in response to female urine was not identical to that in response to male urine. That is, the density of Fos‐ir cells in the caudal portion was slightly larger than that in the rostral portion in the lateral region, while in other regions the density in the rostral portion was higher than that in the caudal portion. It is likely that information from different pheromones is transmitted to the higher brain regions through the different regions of the AOB.

Blockage of urinary responses by inhibitors for IP3-mediated pathway in rat vomeronasal sensory neurons

The mammalian vomeronasal system is involved in the effects of urinary chemicals on gonadal functions and sexual behaviors. For example, exposure to urine affects the timing of oestrous cycles in rats. Rat vomeronasal sensory neurons in slice preparation were studied under on-cell patch clamp conditions. We found that urine excreted from male Wistar rats increased impulse frequency in vomeronasal sensory neurons of female Wistar rats. The urinary responses were blocked by an inositol-1,4,5-trisphosphate (IP3)-channel inhibitor (10 microM ruthenium red) or phospholipase C inhibitors (10 microM U-73122 and 1 mM neomycin), suggesting that pheromone-like substances in the urine induce the response in the rat vomeronasal sensory neurons via the IP3-dependent transduction pathway.

Inositol-1, 4, 5-trisphosphate accumulation induced by urinary pheromones in female rat vomeronasal epithelium

The mechanisms involved in pheromone-induced responses in the vomeronasal neurons, especially in mammals, are still unclear. In the present study, we examined the effects of rat urine samples containing various types of pheromones regulating gonadal functions on the accumulation of cAMP and inositol 1,4,5-trisphosphate (IP3) in a vomeronasal membrane preparation from the female Wistar rat. Stimulation of the preparation with forskolin induced cAMP accumulation, but stimulation with urine samples excreted from the male Wistar rat, the female Wistar rat, and the male Donryu rat did not change cAMP levels. These results were consistent with the electrophysiological results showing that dialysis of a high concentration of cAMP into the vomeronasal neuron does not induce currents. Stimulation with the three urine samples induced the accumulation of IP3 in the membrane preparation. These results are consistent with previous electrophysiological results [K. Inamura, M. Kashiwayanagi, K. Kurihara, Inositol-1,4,5-trisphosphate induces responses in receptor neurons in rat vomeronasal sensory slices, Chem. Senses 22 (1997) 93-103; K. Inamura, M. Kashiwayanagi, K. Kurihara, Blockage of urinary responses by inhibitors for IP3-mediated pathway in rat vomeronasal sensory neurons, Neurosci. Lett. 233 (1997) 129-132]. After the treatment with Pertussis toxin (PTX), the male Wistar urine did not induce IP3 accumulation significantly. Application of the male Wistar urine decreased ADP-ribosylation of Gi with PTX, while that of the male Donryu urine decreased ADP-ribosylation of Go. Thus, the present results support a mechanism by which the responses of the rat vomeronasal neurons to urinary pheromones are mediated by IP3, Gi and/or Go.

Inward current responses to urinary substances in rat vomeronasal sensory neurons

No study has yet demonstrated an inward current in response to pheromonal substances in vomeronasal sensory neurons. Using female rat vomeronasal sensory neurons, we here successfully recorded inward currents in response to urine from various sources. Of the neurons that responded to urine, 77% responded to only one type of urine. Male Wistar urine induced responses preferentially in the apical layer of the sensory epithelium, whilst male Donryu and female Wistar urine induced responses mainly in the basal layer of the epithelium. The amplitude of inward currents induced by application of male Wistar urine was voltage‐dependent with average amplitude of −47.1 ± 6.2 pA at −74 mV. The average reversal potential for male Wistar urine was −9.3 ± 6.1 mV, which was not apparently different from the reversal potentials for urine from different species. It is likely that the urine‐induced inward currents in response to different types of urine are mediated via a similar channel. The simultaneous removal of Na+ and Ca2+ from extracellular solution eliminated the response. The magnitude of the urine‐induced inward current in Cl–‐free external solution was similar to that in normal solution, suggesting that the urine‐induced current is cation selective. Removal of external Ca2+ enhanced the amplitude of the urine‐induced current and prolonged the response. Application of the constant‐field equation indicated a very high permeability coefficient for Ca2+. This study first demonstrated that substances contained in urine elicited inward currents, which induce an excitatory response in vomeronasal sensory neurons, through cation‐selective channels.

Increases in Fos-immunoreactivity after exposure to a combination of two male urinary components in the accessory olfactory bulb of the female rat.

Exposure to either the dialyzed urine preparation (<500 Da) or the remaining substances (>500 Da) did not induce expression of Fos-immunoreactive cells in the mitral/tufted cell layer of the accessory olfactory bulb (AOB), whereas exposure to a mixture of these preparation did induce expression. These results suggest that a combination of low and high molecular weight substances is necessary for the increases in Fos-immunoreactivity in the AOB.

Changes in Fos expression in the accessory olfactory bulb of sexually experienced male rats after exposure to female urinary pheromones.

We studied Fos‐immunoreactive (Fos‐ir) structures in the accessory olfactory bulb (AOB) of rats after the vomeronasal organ was exposed to urine. Exposure of the vomeronasal organ of male Wistar rats to oestrous and dioestrous female Wistar urine led to the appearance of many Fos‐ir cells in the rostral region of the periglomerular cell (PGC) layer, but induced few Fos‐ir cells in the caudal region. These results suggest that the regionalization of Fos‐ir cells after exposure to female urine is remarkable in the PGC layer of the AOB. Sexually experienced male rats have been shown to prefer oestrous to dioestrous female urine, while sexually inexperienced males do not exhibit these preferences. In the present study, we compared the expression of Fos‐ir cells in the AOB of sexually experienced and sexually inexperienced male rats following exposure to oestrous and dioestrous urine. In the localized region (lateral and rostral sectors) of the PGC layer, many more Fos‐ir cells were expressed in the sexually experienced rats than in the inexperienced rats. These results suggest that sexual experience in males enhances the transmission of reproductively salient information concerning potential oestrous status to a specific PGC region of the AOB.

Cluster of proliferating cells in rat vomeronasal sensory epithelium.

We investigated the properties of small cells in the vomeronasal sensory epithelium of adult rats. The sensory neurons in the sensory epithelium were stained by antibodies to Gi2α and Goα in their cell bodies and dendrites, while the small cells, which formed a cluster in the epithelium, were not stained at all. Voltage-activated inward currents were not detected by patch-clamp recordings, but outward currents were induced by the application of voltage step pulses. These results suggest that the small cells are different from the vomeronasal sensory neurons. Bromodeoxyuridine (BrdU) labeling indicated that dividing cells existed in the cluster of small cells.

Effects of cGMP and sodium nitroprusside on odor responses in turtle olfactory sensory neurons

The effects of cGMP and sodium nitroprusside (SNP) on odor responses in isolated turtle olfactory neurons were examined. The inward current induced by dialysis of a mixture of 1 mM cAMP and 1 mM cGMP was similar to that induced by dialysis of 1 mM cAMP or 1 mM cGMP alone. After the neurons were desensitized by the application of 1 mM cGMP, 3 mM 8-(4-chlorophenylthio)-cAMP, a membrane-permeable cAMP analog, did not elicit any current, indicating that both cAMP and cGMP activated the same channel. Extracellular application of SNP, a nitric oxide (NO) donor, evoked inward currents in a dose-dependent manner. However, application of SNP did not induce any currents after desensitization of the cGMP-induced currents, suggesting that SNP-induced currents are mediated via the cGMP-dependent pathway. Application of the cAMP-producing odorants to the neurons induced a large inward current even after neurons were desensitized to a high concentration of cGMP or SNP. These results suggest that the transduction pathway independent of cAMP, cGMP, and NO also contributes to the generation of odor responses in addition to the cAMP-dependent pathway.

Odor discrimination of "IP3-" and “cAMP-increasing” odorants in the turtle olfactory bulb

The ability of the turtle olfactory system to discriminate between various odorants that increase levels of adenosine 3′,5′-cyclic monophosphate (cAMP) and mositol trisphosphate (IP3) in the olfactory bulb was examined by the cross-adaptation technique and analyzed by multidimensional scaling. The mean values of the degree of discrimination among the IP3-increasing odorants were higher than those among the cAMP-increasing odorants, and were similar to those between cAMP- and IP3-increasing odorants, suggesting that the features of the receptors of cAMP-increasing odorants are different from those which respond to IP3-increasing odorants. Analysis by multidimensional scaling suggested that differences in second messenger pathways are not related to detecting odor quality in the turtle olfactory system.